Transmission of Sugarcane White Leaf Phytoplasma by Yamatotettix flavovittatus, a New Vector Author(s): Y. Hanboonsong, W. Ritthison, C. Choosai, and P. Sirithorn Source: Journal of Economic Entomology, 99(5):1531-1537. 2006. Published By: Entomological Society of America DOI: http://dx.doi.org/10.1603/0022-0493-99.5.1531 URL: http://www.bioone.org/doi/full/10.1603/0022-0493-99.5.1531

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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. FORUM Transmission of Sugarcane White Leaf Phytoplasma by Yamatotettix flavovittatus, a New Leafhopper Vector

1,2 1 1 3 Y. HANBOONSONG, W. RITTHISON, C. CHOOSAI, AND P. SIRITHORN

J. Econ. Entomol. 99(5): 1531Ð1537 (2006) ABSTRACT Sugarcane white leaf disease is caused by plant pathogenic phytoplasmas that are transmitted to the plant by the leafhopper Matsumuratettix hiroglyphicus (Matsumura). To determine whether there are other vectors that transmit this disease pathogen, leafhopper species in sugarcane, Saccharum officinarum L., Þelds in northeastern Thailand were monitored by using light traps. Sixty-nine leafhopper species from family Cicadellidae were found. Using nested polymerase chain reaction (PCR) with speciÞc primers, a 210-bp ampliÞed DNA fragment corresponding to phytoplasma associated with sugarcane white leaf disease was detected from 12 species of [Balclutha rubrostriata (Melichar), Balclutha sp., Bhatia olivacea (Melichar), Exitianus indicus Distant, Macrosteles striifrons Anufriew, Matsumuratettix hiroglyphicus (Matsumura), Recilia distincta (Mot- schulsky), Recilia dorsalis (Motschulsky), Recilia sp., Thaia oryzivora Ghauri, Yamatotettix flavovittatus Matsumura, and Xestocephalus sp.]. The percentage of individual infection with phytoplasma varied from 5% in B. olivacea to 35% in Xestocephalus sp. The most abundant leafhopper species, i.e., E. indicus, Y. flavovittatus, and M. hiroglyphicus were used in transmission tests to determine their vector status for the sugarcane white leaf phytoplasma transmission. Infected were reared on healthy plants and speciÞc PCR followed by sequencing of the amplicons was used to determine whether the phytoplasma was transmitted to the plants. The results showed that both Y. flavovittatus and M. hiroglyphicus, but not E. indicus, can transmit sugarcane white leaf phytoplasma to healthy sugarcane plants. The transmission efÞciency of M. hiroglyphicus (55%) was higher than that of Y. flavovittatus (45%). We conclude that Y. flavovittatus is a newly discovered vector for sugarcane white leaf disease, in addition to M. hiroglyphicus. These two species peak at different times of the year and therefore complement each other in the transmission of the phytoplasma. Because there are no known alternative host plants for the sugarcane white leaf, management of the disease will necessarily require the control of both Y. flavovittatus and M. hiroglyphicus.

KEY WORDS Matsumuratettix hiroglyphicus, Yamatotettix flavovittatus, phytoplasma, sugarcane white leaf disease, transmission

Sugarcane white leaf (SCWL) disease is one of the ceeded. Attempts to Þnd alternative host plants were most destructive diseases of sugarcane, Saccharum of- made, and it was thought for many years that the ficinarum L., in Thailand. This disease is caused by a gramineous weeds such as Bermuda grass, Cynodon phytoplasma, which produces typical symptoms of dactylon (L.); crowfoot grass, Dactyloctenium aegyp- total leaf chlorosis and tiller proliferation (Nakashima tium (L.) Willd.; and brachiaria grass, Brachiaria dis- et al. 1994, Wongkaew et al. 1997). Sequences ob- tachya (L.) Stapf., also were reservoirs for the phyto- tained from the intergenic spacer region between the plasma. However, sequencing data showed that the 16S and the 23S rDNA showed that the SCWL patho- phytoplasma that infect these weeds were different gen belongs to the 16SrXI-B genetic group (Cronje et from those infecting sugarcane (Hanboonsong et al. al. 1998, Lee et al. 1998). This phytoplasma cannot be 2002). It is known that phytoplasmas that cause plant cultured and is an obligate parasite of the host cell. diseases are transmitted and spread by phloem-feed- Apart from disease management by eradication of ing insects, most commonly leafhoppers (Membra- infected plants to decrease the amount of inoculum, coidea) but also some planthoppers (Fulgoroidea) no efÞcient method of disease control has yet suc- and psyllids (Psylloidea) (Ploaie 1981, Hill and Sin- clair 2000). In sugarcane disease, the leafhopper Mat- sumuratettix hiroglyphicus (Matsumura) in the subor- 1 Department of Entomology, Faculty of Agriculture, Khon Kaen der Auchenorrhyncha of the is the University, Khon Kaen 40002, Thailand. only insect known to transmit SCWL phytoplasma in 2 _ _ Corresponding author, e-mail: yupa [email protected] or yupa Taiwan and Thailand (Matsumoto et al. 1968, Chen [email protected]. 3 Department of Plant Pathology, Faculty of Agriculture, Khon 1974, Hanboonsong et al. 2002). It was reported that Kaen University, Khon Kaen 40002, Thailand. the leafhopper vector also plays an important role as

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a reservoir of the SCWL phytoplasma. The phyto- insects from each species were then transferred in plasma is widely distributed throughout the body of groups of Þve on a total of 20 test plants (3-wk old the insect and showed transovarial transmission from disease-free sugarcane seedlings) for 5 d. After the generation to generation of the insect vector (Han- inoculation access period, test plants were maintained boonsong et al. 2002). Thus, the leafhopper M. hiro- in nylon mesh insect-proof cages and kept in an insect- glyphicus is a key target for SCWL control, because it proof greenhouse for 8 wk until PCR assays. Symptoms is both reservoir and vector for the phytoplasma. It is of disease development also were observed daily. known that several species of leafhoppers can trans- Twenty individual plants with no contact with insects mit the same kind of plant phytoplasma and that a kept in the same condition as the test plants were used particular leafhopper species is also able to transmit as control. several phytoplasma pathogens (Fletcher et al. 1998, PCR Assays for SCWL Phytoplasma in Test Plants. Lee et al. 2000, Palmano and Firrao 2000). In this DNA from each test plant and SCWL disease plants study, we set out to determine whether there are was extracted from two leaf midribs by using the other leafhopper species that transmit the sugarcane cetyltrimethylammonium bromide method (Kollar et white leaf phytoplasma in addition to M. hiroglyphicus. al. 1990, Nakashima et al. 1991). PCR ampliÞcation was performed in a 25-␮l reaction mixture containing 20Ð25 ng of plant genomic DNA, 0.2 mM each dNTP, Materials and Methods 0.25 ␮M each primer,1UofTaqDNA polymerase (Promega, Madison WI) in 1ϫ PCR reaction buffer Plant Sources. White leaf disease and healthy sug- (supplied by the manufacturer) containing 1.5 mM arcane plants from 8 mo old were collected from MgCl2. Two sets of oligonucleotide primers matching sugarcane-growing areas in Udonthani Province, the 16S rRNA and 23S rRNA of phytoplasmas were northeastern Thailand. The axillary buds from both used (Namba et al. 1993, Wongkaew et al. 1997). ends of healthy sugarcane shoots were tested by poly- Primers MLO-X and MLO-Y were used in the Þrst merase chain reaction (PCR) with speciÞc SCWL round PCR, and primers P1 and P2 were used in nested primers to conÞrm their disease-free status. Then, PCR (Wongkaew et al. 1997, Hanboonsong et al. young healthy sugarcane plants were generated from 2002). PCR conditions were as follows: denaturation the remaining axillary buds of the middle part of the for 1 min (5 min for the Þrst cycle) at 92ЊC, annealing shoot. The plants were grown in pots and kept in an temperature of 60ЊC for 30 s, and an extension time of insect-proof greenhouse. When the plants were 3 wk 1.30 min (10 min for the last cycle) at 72ЊC for 35 old, before using them for the transmission test, a few cycles. The nested-PCR assays were carried out using leafs were tested by PCR to conÞrm their disease-free 1 ␮l of the Þrst PCR product (diluted 1:50 in sterile status. Monitoring and Collection of Leafhoppers. Leaf- deionized water) as template. In total, 40 cycles were hoppers from sugarcane Þelds in Udonthani Province conducted for nested PCR under the same conditions of denaturation and extension as the Þrst PCR, except were collected monthly by using light traps to attract Њ the insects and an insect aspirator to catch them. The that the annealing temperature was 68 C. Test plants Þrst insects captured were sent to a leafhopper tax- without contact with insects and no DNA were used onomist in Japan for species identiÞcation. Additional as negative controls. DNA from a SCWL plant was specimens were identiÞed by comparison to the used as positive control. PCR products were analyzed voucher specimen identiÞed by the leafhopper tax- by electrophoresis on a 1.5% agarose gel in Tris borate- ␮ onomist. The leafhoppers were counted and tested for EDTA buffer containing 0.5 g/ml ethidium bromide. SCWL phytoplasma by speciÞc PCR. Occurrence of PCR Assays for SCWL Phytoplasma in Leafhop- white leaf disease in the sugarcane Þeld also was sur- pers. Adults leafhoppers collected from sugarcane veyed monthly from seedling (2 mo old) until har- Þelds were ground singly in Eppendorf tubes with vesting stages (11Ð12 mo old). The disease survey was DNA extraction buffer (200 mM Tris, pH 8.0, 250 mM set by observing and counting plants showing disease NaCl, 25 mM EDTA, 0.5% SDS, and 0.1 mg/ml pro- symptoms from 130 plants per row. A total of 12 rows teinase K). Insect DNA was extracted by the phenol- with a 10-plant interval between rows from 2.1 ha were chloroform method (Hanboonsong et al. 2002). PCR monitored. assays for SCWL phytoplasma in insect leafhoppers Transmission Test. Three leafhoppers species Mat- were carried out as described for the test plants. sumuratettix hiroglyphicus (Matsumura), Yamatotettix Sequencing Analysis. The 210-bp products of the flavovittatus Matsumura, and Exitianus indicus Distant nested PCR ampliÞcation of test plants and two leaf- were collected from sugarcane Þelds and maintained hoppers species (M. hiroglyphicus and Y. flavovittatus) separately on disease-free young sugarcane plants in were puriÞed using a commercial kit (QIAquick PCR nylon mesh insect-proof cages. Then, plants and insect puriÞcation kit, QIAGEN, Valencia, CA) and se- colonies were kept in an insect-proof greenhouse. quenced using an ABI Prism automated sequencer Insects were kept without food for 4 h before the (Applied Biosystems, Foster City, CA). The sequenc- transmission test. Then, thirty individuals of each in- ing data from both sugarcane plants and leafhoppers sect species were placed on caged SCWL symptomatic were aligned and analyzed by using SeqMan, version plants for acquisition periods of 48 h. One hundred 5.08 (DNASTAR Inc., Madison, WI). October 2006 HANBOONSONG ET AL.: LEAFHOPPER TRANSMISSION OF SCWL PHYTOPLASMA 1533

Table 1. Leafhopper species of family Cicadellidae captured product of 210 bp speciÞc for phytoplasma associated from sugarcane fields with SCWL was detected in 12 of 69 Þeld-collected leafhopper species (Table 2). The percentage of in- No. captured Subfamily Species names insects in dividual insects infected with the phytoplasma varied 2002Ð2003 from 5% [Bhatia olivacea (Melichar)] to 35% (Xesto- Cicadellinae Cicadella spp. 25 cephalus sp.). In comparison, the known leafhopper Cofana spectra (Distant) 22 vector M. hiroglyphicus showed 25% of phytoplasma Cofana subvirescens (Stal.) 12 infection. Deltocephalinae Balclutha spp. 294 Transmission of SCWL Phytoplasma from Leaf- Balclutha rubrostriata (Melichar) 33 spp. 10 hoppers to Plants. The three most abundant leafhop- Exitianus indicus Distant 942 per species that were positive for SCWL phytoplasma Exitianus spp. 35 (M. hiroglyphicus, E. indicus, and Y. flavovittatus) were Hecalus arcuatus (Motschulsky) 4 used for the transmission trial. After 8 wk of incuba- Hecalus prasinus Matsumura 31 Hecalus porrectus (Walker) 7 tion, none of the sugarcane plants fed on by the three Macrosteles striifrons Anufriew. 105 leafhopper species showed any white leaf symptoms. Matsumuratettix hiroglyphicus 1,362 However, when the nested PCR with speciÞc SCWL (Matsumura) phytoplasma primers was used on these plants, a Nephotettix nigropictus (Stal.) 13 Nephotettix pravus Matsumura 33 210-bp SCWL phytoplasma-speciÞc band was ob- Nephotettix virescens (Distant) 26 tained in 55 and 45% of those plants that were used to Paralimnus sp. 6 feed M. hiroglyphicus and Y. flavovittatus, respectively Recilia spp. 419 (Table 3). Thus, M. hiroglyphicus and Y. flavovittatus Recilia distincta (Motschulsky) 299 Recilia dorsalis (Motschulsky) 148 can transmit sugarcane white leaf phytoplasma to Scaphoideus festivus Matsumura 16 healthy sugarcane plants. No phytoplasma was de- Scaphoideus spp. 13 tected in the sugarcane plants fed to E. indicus (Fig. 2) Yamatotettix flavovittatus 431 and in control plants that were not exposed to leaf- Matsumura Selenocephalinae Bhatia olivacea (Melichar) 156 hoppers. Typhlocybinae Empoascanara spp. 76 Sequencing Data. To conÞrm that the phytoplasma Thaia oryzivora Ghauri 72 ampliÞed from the plants infected in the laboratory Xestocephalinae Xestocephalus spp. 170 was the same as the phytoplasma that infects the Þeld-collected plants, we sequenced the PCR ampli- cons with the primers that were used for the nested Results PCR. We found that the sequences of hypervariable Distribution of Leafhoppers in Sugarcane-Growing 16S/23S DNA spacer region obtained from Y. flavo- Area. Leafhopper insects from the sugarcane-growing vittatus infected in the laboratory, M. hiroglyphicus areas were trapped monthly by using light traps as infected in the laboratory, sugarcane collected in the described. Insects from 16 genera of family Cicadel- Þeld, sugarcane infected in the laboratory with lidae were captured and identiÞed (Table 1). The M. hiroglyphicus, and sugarcane infected in the labo- leafhoppers M. hiroglyphicus, E. indicus, and Y. fla- ratory with Y. flavovittatus were identical (Fig. 3). In vovittatus were the most abundant species recorded. contrast, there were several differences between Population dynamics studies of leafhoppers showed these sequences and those obtained from sugarcane high populations during the rainy season from late with grassy shoot disease (another sugarcane disease April to August (Fig. 1). However, the highest peak caused by phytoplasma) or from the weed Bermuda period varied with species. For example, the peaks for grass, Cynodon dactylon (L.), with white leaf disease. M. hiroglyphicus and E. indicus were during AprilÑ May, whereas those for Y. flavovittatus were JulyÐ Discussion August. These population dynamics results were ob- tained in 2002Ð2003 and are consistent with those SCWL disease is a major agricultural problem, obtained at the same location in 1997Ð1999 (Wong- causing severe losses to the sugarcane industry. Ef- kaew 1999). A correlation between abundance of leaf- fective control of the disease requires a good under- hoppers trapped and disease prevalence was not al- standing of the vector and reservoir of the phyto- ways present. The highest prevalence of SCWL plasma. It has been known for many years that the disease was in AugustÐSeptember, which corresponds leafhopper M. hiroglyphicus is a vector of the disease, to the population peak of Y. flavovittatus but is several and we have shown in recently published work that months later than the peak of M. hiroglyphicus. The this insect is also the reservoir, and not the weeds that highest disease prevalence was when the sugarcane grow in sugarcane Þelds, as previously thought. We was at the elongation phase in August to September therefore set out to determine whether other species rather than in the tillering and harvesting phases of leafhopper also were implicated in the transmission (Fig. 1). of SCWL phytoplasma to the sugarcane, based on the Testing Insects for Infection with SCWL Phyto- following criteria: 1) the insects must be abundant in plasma. Nested PCR with primers speciÞc for SCWL the sugarcane-growing areas; 2) their populations dy- phytoplasma was used to screen Þeld-collected leaf- namics must match the cycle of the SCWL disease, i.e., hoppers for potential vectors of the disease. A PCR they must peak when the disease is most prevalent; 1534 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 99, no. 5

Fig. 1. Abundance and population dynamics of three leafhoppers species (M. hiroglyphicus, E. indicus, and Y. flavovittatus) and percentage of symptomatic sugarcane with white leaf disease

3) They must carry the phytoplasma; and 4) they must from infected plant to insect to healthy plant was infect healthy plants in the transmission test. Although demonstrated only with Y. flavovittatus. This insect is other criteria also could have been used instead or in fairly abundant, and its population shows a peak in addition to criteria 1 and 2 (e.g., a low-abundance, JulyÐAugust, closely matching the period of highest highly active vector may be very effective in spreading incidence of SCWL disease (AugustÐSeptember). a disease), our criteria were easily measurable and In the transmission test, we demonstrated infec- useful for an initial screen. tion by PCR ampliÞcation of the phytoplasma from Using criteria 1Ð3, we limited the number of can- the sugarcane, but we did not observe any symptoms didate species to three, down from 69 species. These of disease. This is consistent with previous studies of species were M. hiroglyphicus, the known vector of infection by phytoplasma of sugarcane and other plants SCWL disease, which demonstrated the validity of our where it was found that disease symptoms rarely occur criteria. The two other species were Y. flavovittatus in the laboratory (Tran-Nguyen et al. 2000, Pilkington et and E. indicus, which were subjected to the transmis- al. 2004). Besides, the rates of phytoplasma disease sion test. Transmission of the SCWL phytoplasma symptom expression in test plants are often reported to be low. Jarausch et al. (2001) reported 2% (one of 50 Table 2. Percentage of individuals infected with SCWL phy- test plants) symptom transmission success between toplasma from 12 leafhopper species detected by nested PCR psyllid Cacopsylla pruni (Scopoli) and apricot seed- lings infected with European stone fruit yellow phy- Leafhopper species % infection toplasma. A 3.5% (three of 88 test plants) rate of Xestocephalus sp. 34.62 B. rubrostriata (Melichar) 30.76 Thaia oryzivora Ghauri 30.00 Table 3. Percentage of transmission of SCWL phytoplasma by M. hiroglyphicus 25.35 three leafhoppers species to healthy sugarcane plants Balclutha sp. 23.73 Y. flavovittatus 15.68 No. infected plants/ Leafhopper species % transmission Recilia distincta (Motschulsky) 13.21 no. of test plants Recilia dorsalis (Motschulsky) 12.96 E. indicus 12.31 M. hiroglyphicus 11/20 55 Recilia sp. 10.00 Y. flavovittatus 9/20 45 M. striifrons Anufriew 9.52 E. indicus 0/20 0 B. olivacea 5.41 Control (no insect) 0/20 0 October 2006 HANBOONSONG ET AL.: LEAFHOPPER TRANSMISSION OF SCWL PHYTOPLASMA 1535

The prevalence of Y. flavovittatus in the sugarcane Þelds is very similar to that of M. hiroglyphicus and its infection rate in the laboratory is somewhat lower (45 versus 55%). Furthermore, the population peaks of M. hiroglyphicus and of Y. flavovittatus follow each other, hardly overlap, and together cover the period of highest prevalence of the SCWL disease. The results of the population dynamics suggest that M. hiroglyphi- cus transmits the SCWL phytoplasma preferentially to the plants when they are at early elongation stage, whereas Y. flavovittatus transmits preferentially to late Fig. 2. Agarose gel electrophoresis of test plant DNA elongation stage plants. This might explain why the ampliÞed by nested PCR with primers MLO-X/MLO-Y fol- percentage of infectivity of Y. flavovittatus was some- lowed by primers P1 and P2. N is no DNA sample (negative what low, because we used very young plants in the control), and P is sugarcane white leaf DNA (positive con- trol). Samples in lanes 1 and 2 are DNA from sugarcane transmission test. Thus, it seems that M. hiroglyphicus healthy plant, lanes 3Ð5 are DNA from a sugarcane plant with and Y. flavovittatus are equally important in the trans- white leaf disease symptoms collected from the Þeld, lanes mission of SCWL phytoplasma. 6Ð9 are DNA from plants infected with leafhopper M. hiro- There are many reports in the literature of multiple glyphicus, lanes 10Ð13 are DNA from plants infected with leafhopper species transmitting the same phyto- leafhopper Y. flavovittatus, and M is the 100-bp ladder (sizes plasma. For example, chrysanthemum yellows phyto- are shown in base pairs) plasma has been reported to be transmitted by three leafhopper species: Euscelidius variegatus Kirsch- successful transmission of symptom was reported in baum, Macrosteles quadripunctulatus (Kirschbaum), trials between leafhopper Oncopsis alni (Schrank) and and Euscelis incisus (Kirschbaum) (Domenico et al. grapevine yellow symptoms (Maixner et al. 2000). 1997). Aster yellows phytoplasma is transmitted by 24 However, a high transmission rate of 66% (24 of 36 test leafhoppers species, and 15 leafhoppers species can plants) also was reported in transmission tests with transmit peach-X disease phytoplasma (Lee et al. sugarcane yellow leaf phytoplasma by planthopper 1998). Saccharosydne saccharivora (Westwood) (Arocha et Despite evidence of being infected by phytoplasma, al. 2005). In our studies, the test plants did not show E. indicus did not transmit the SCWL phytoplasma to any disease symptoms. This could be due to a low the plants. This is not surprising, because the presence phytoplasma titer in the relatively short period in of the phytoplasma in the insect body does not nec- which we observed the plants (8 wk). Growing of the essary mean that the insect is a vector for a disease test plants used in the transmission test in pots may (Vega et al. 1993). Our study identiÞed 12 species of have effected the normal development of symptoms of leafhopper present in sugarcane Þelds that carry the disease. The sugarcane cultivar used in the trans- SCWL phytoplasma, the most abundant three species mission test also is known to be one of the major of which were used for further tests. It is noteworthy factors inßuencing symptom appearance (Cronje et that 3% of the sugarcane plants showed symptoms of al. 1998, Arocha et al. 2005). Furthermore, it has been SCWL disease in March, but no M. hiroglyphicus or suggested that symptoms of the disease occur only in Y. flavovittatus insects were collected from January to certain environmental conditions, such as water short- March. It is therefore possible that some of the less age or high numbers of defoliating insects (Bertaccini abundant nine species also are capable of transmit- et al. 1996, Tran-Nguyen et al. 2000). It is noteworthy tingthephytoplasmatotheplants.Forexample,Macro- that when we conducted the SCWL phytoplasma steles striifrons Anfriew. which is a polyphagous leaf- transmission test with M. hiroglyphicus, the plants also hopper species and one of the 12 PCR-positive species, did not show any symptoms of the disease (data not has been previously shown to be a vector for 16 SrI-B shown). phytoplasma group causing several phytoplasma Because relying on disease symptoms in plants in- diseases of eggplant dwarf and tomato and marguerite fected in the laboratory may lead to false negatives, yellows (Lee et al. 1998). Future studies will deter- we relied on the PCR test, and we sequenced the mine whether there are other vectors of SCWL products of PCR ampliÞcation of sugarcane phyto- disease among the species that we have collected. plasma DNA obtained from the transmission tests and Because the alternative host plants for the SCWL compared them to that of infected plants collected phytoplasma are unknown, understanding of the in- from Þelds. The 16S/26S spacer region sequenced is sect vectorsÕ ecology is of prime importance. Our Þnd- hypervariable (it is not under the same evolutionary ing that Y. flavovittatus is also a vector for SCWL pressure as the ribosomal RNA structural genes) and disease, in addition to previously known leafhopper has been used previously to distinguish different phy- vector M. hiroglyphicus, and that it peaks after toplasmas (Kirkpatrick and Smart 1995, Hanboonsong M. hiroglyphicus explains the disease incidence after et al. 2002). We found a perfect match between the the seasonal decline of the M. hiroglyphicus popula- sequences obtained, demonstrating that the species of tion. Therefore, the disease can occur continuously phytoplasma transmitted by Y. flavovittatus is the over time. Further studies of this insect biology, ecol- agent of SCWL disease. ogy, and phytoplasma relationships can be used to 1536 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 99, no. 5

Fig. 3. Alignment of DNA sequences from the spacer region of phytoplasma DNA ampliÞed from plants infected with leafhoppers M. hiroglyphicus (M), Y. flavovittatus (Y), and compared with the sequence of DNA ampliÞed from sugarcane with white leaf disease (SCWL), insect leafhoppers M. hiroglyphicus (IM) and Y. flavovittatus (IY), sugarcane with grassy shoot disease (gr sh), and Bermuda grass with white leaf disease (Berm). The sequences of M, Y, SCWL, IM, and IY are identical but different from those of gr sh and Berm. establish potential SCWL disease management strat- Bertaccini, A., L. Mittempergher, and M. Vibio. 1996. Iden- egies. tiÞcation of phytoplasmas associated with a decline of European hackberry (Celtis australis). Ann. Appl. Biol. 128: 245Ð253. Acknowledgments Chen, C. T. 1974. Sugarcane white leaf disease in Thailand We thank Dr. Masami Hayashi (Saitama University, and Taiwan. Sugarcane Pathol. Newsl. 11/12: 23. Saitama City, Saitama, Japan) for leafhopper identiÞcation, Cronje, C.P.R., A. M. Tymon, P. Jones, and R. A. Bailey. 1998. Upsorn Pliansinchai (Mitr Phol Sugar Co., Ltd., Klongtoey, Association of a phytoplasma with a yellow leaf syndrome Bangkok) for providing the sugarcane stocks, and Dr. As- of sugarcane in Africa. Ann. Appl. Biol. 133: 177Ð186. sanee Pachinburavan (Faculty of Agriculture, Khon Kaen Domenico, B., M. Claudia, and B. Guido. 1997. Differential University, Khon Kaen, Thailand) for critical review of the acquisition of chrysanthemum yellows phytoplasma by manuscript. This work was funded by the National Research three leafhopper species. Entomol. Exp. Appl. 83: 219Ð224. Council of Thailand and Khon Kaen University. Fletcher, F., A. Wayadande, U. Melcher, and F. Ye. 1998. The phytopathogenic mollicute-insect vector interface: a closer look. Phytopathology 88: 1351Ð1358. References Cited Hanboonsong, Y., C. Choosai, S. Panyim, and S. Damak. Arocha, Y., M. Lopez, M. Fernandez, B. Pinol, D. Horta, 2002. Transovarial transmission of sugarcane white leaf E. L. Peralta, R. Almeida, O. Carvajal, S. Picornell, phytoplasma in the insect vector Matsumuratettix hiro- M. R. Wilson, et al. 2005. Transmission of a sugarcane glyphicus (Matsumura). Insect Mol. Biol. 11: 97Ð103. yellow leaf phytoplasma by the delphacid planthopper Hill, G. T., and W. A. Sinclair. 2000. Taxa of leafhoppers Saccharosydne saccharivora, a new vector of sugarcane carrying phytoplamsmas at sites of ash yellows occur- yellow leaf syndrome. J. Plant Pathol. 54: 634Ð642. rence in New York state. Plant Dis. 84: 134Ð138. October 2006 HANBOONSONG ET AL.: LEAFHOPPER TRANSMISSION OF SCWL PHYTOPLASMA 1537

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